A method for constructing a characteristic spectrum of Antai ointment and its application

Abstract

The present invention relates to the technical field of traditional Chinese medicine analysis, and more particularly to a method for constructing a characteristic spectrum of Antai Ointment and its application. The method comprises the following steps: 1. preparing a reference solution; 2. preparing a test solution; 3. determining measurement conditions; and 4. importing the chromatogram into a traditional Chinese medicine spectrum to generate a control characteristic spectrum. This characteristic method identifies five characteristic peaks, has good repeatability and high precision, and comprehensively and comprehensively displays the chemical composition characteristics of Antai Ointment, thereby achieving holistic control of the pharmaceutical quality of Antai Ointment.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis, and in particular to a method for constructing a characteristic spectrum of Antai ointment and an application thereof. Background Art

[0002] Antai Ointment, listed in the 2020 edition of the Chinese Pharmacopoeia (Volume 1), is an ointment formulated from three medicinal herbs: Sanguisorba officinalis charcoal, Chinese gallnut, and borneol, along with two APIs: berberine hydrochloride and papaverine hydrochloride. It boasts cooling blood and stopping bleeding, clearing heat and detoxifying, drying dampness and healing sores, and relieving swelling and pain. Pharmacopoeia standards include two thin-layer chromatography (TLC) assays and content determination. Currently, patents and literature lack a characterization profile for Antai Ointment, making it difficult to assess its overall quality.

[0003] Difficulties in constructing the characteristic map of Antai Ointment:

[0004] (1) The complexity of the sample makes it difficult to prepare and purify the active ingredients and remove impurity interference: Complex samples such as traditional Chinese medicine contain multiple components with different polarity, molecular weight and chemical properties, which increases the difficulty of separation and detection;

[0005] (2) It is difficult to select a high-precision detection method: Selecting an appropriate analytical method is the key to preparing a characteristic spectrum; commonly used analytical methods include high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UPLC), etc.; however, different methods have different sample processing and detection capabilities. It is necessary to select the most appropriate method based on the characteristics of the sample to improve the accuracy of the analysis and detection;

[0006] (3) Standardization issues: In order to ensure the comparability and reproducibility of characteristic spectra, it is necessary to establish standardized preparation and analysis processes; this includes sample pretreatment, selection of chromatographic conditions, data processing methods, etc. The lack of standardization may lead to inconsistent results between different laboratories;

[0007] (4) Identification and attribution of characteristic peaks: In the characteristic spectrum, characteristic peaks need to be accurately identified and attributed; this requires an in-depth understanding of the chemical properties and chromatographic behavior of each component in the sample. Incorrect identification and attribution of characteristic peaks may lead to inaccurate characteristic spectra. Summary of the Invention

[0008] One of the purposes of the present invention is to provide a method for constructing a characteristic spectrum of An Tai Ointment, which can comprehensively, systematically and accurately control and evaluate the pharmaceutical quality of An Tai Ointment.

[0009] The second purpose of the present invention is to apply the characteristic spectrum method provided by the present invention to the overall control and evaluation of the pharmaceutical quality of An Tai Ointment.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0011] A method for constructing a characteristic spectrum of Antai ointment comprises the following steps:

[0012] (1) Preparation of reference solution: Take the reference substance, weigh it accurately, add methanol to make a solution containing 60 μg per 1 ml, shake well, and use it as the reference solution; the reference substances are: gallic acid, ellagic acid, and berberine hydrochloride;

[0013] (2) Preparation of test solution: Take 5 g of this product, add 50 ml of methanol: water: n-butyric acid mixed solution with a volume ratio of 1:1:0.05, ultrasonically treat for 30-50 min, centrifuge, take the supernatant and evaporate to dryness, dissolve the evaporated material in 30 ml of water, then add petroleum ether to extract 3 times, 30 ml each time, discard the petroleum ether liquid, extract the water liquid with n-butanol 3 times, 30 ml each time, evaporate the n-butanol liquid, add 5 ml of methanol to the material evaporated to dryness, pass through a 0.45 μm microporous filter membrane, discard the initial filtrate, and collect the subsequent filtrate as the test solution.

[0014] (3) Determination: Inject the reference solution and the test solution into a high performance liquid chromatograph according to the following chromatographic conditions, inject 10 μl of each sample, and record the chromatogram;

[0015] Chromatographic conditions were as follows: octadecylsilane bonded silica gel as the filler; acetonitrile-methanol (volume ratio: 27:73) as the mobile phase A, sodium octane sulfonate-triethylamine-phosphoric acid solution as the mobile phase B, elution according to the specified gradient; column temperature: 30°C; flow rate: 1 ml / min; UV detection wavelength: 250-270 nm;

[0016] The elution gradient conditions were:

[0017] From 0 to 30 min, the volume ratio of mobile phase A:phase B changed from 5:95 to 40:60;

[0018] From 30 to 45 min, the volume ratio of mobile phase A:phase B was changed from 40:60 to 70:30;

[0019] From 45 to 48 minutes, the volume ratio of mobile phase A:phase B was changed from 70:30 to 5:95;

[0020] 48-60 min, mobile phase A:B, volume ratio 5:95;

[0021] (4) Generate a control characteristic spectrum: Select the chromatographic peaks that exist in the chromatograms of different batches of An Tai Ointment as common peaks, and use the average value calculation method to generate a control characteristic spectrum of An Tai Ointment.

[0022] Preferably, the sodium octane sulfonate-triethylamine-phosphoric acid solution in step (3) is prepared as follows: 2.4 g of sodium octane sulfonate is dissolved in 1000 ml of water, 4 ml of triethylamine is added, the mixture is shaken, and the pH value is adjusted to 3.0 with phosphoric acid to obtain a sodium octane sulfonate-triethylamine-phosphoric acid solution.

[0023] Preferably, the chromatographic column in step (3) is: Inertsustain-C18 4.6x250mm, 5um.

[0024] Preferably, the detection wavelength of the ultraviolet detector in step (3) is 250 nm.

[0025] Preferably, the control characteristic spectrum generated in step (4) includes five common chromatographic peaks, namely: Peak 1 corresponding to gallic acid, Peak 3 corresponding to ellagic acid, and Peak 5 corresponding to berberine hydrochloride; the characteristic peaks are based on the ellagic acid chromatographic peak as a reference peak, and the relative retention times of the five chromatographic peaks are: Peak 1 is 0.33, Peak 2 is 0.80, Peak 4 is 1.22, and Peak 5 is 1.49.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention established an HPLC characteristic spectrum for Antai ointment, identified five characteristic peaks, and more fully and comprehensively displayed the chemical composition characteristics of Antai ointment; and the sample pretreatment of the present invention is simple, easy to operate, and fast, and the sample extraction solvent, solvent ratio and extraction time, mobile phase type and mobile phase ratio were investigated. Within the investigation range, a methanol: water: n-butyric acid mixed solution with a volume ratio of 1:1:0.05 was preferably used as the extraction solvent, acetonitrile-methanol (27:73) was preferably used as the mobile phase A, 2.4 g of sodium octane sulfonate was dissolved in 1000 ml of water, 4 ml of triethylamine was added, and the mixture was shaken, and the pH value was adjusted to 3.0 with phosphoric acid to obtain mobile phase B as the mobile phase;

[0028] (2) The method for the characteristic spectrum of Antai Ointment constructed in the present invention was methodologically validated, including specificity, precision, repeatability, and stability experiments. The RSD values ​​of the relative retention time of each peak in each experimental result were all ≤1.0%, and the RSD values ​​of the relative peak area were all ≤1.0%. This shows that the characteristic spectrum method is good and can reflect the major chemical components in Antai Ointment. The overall quality of Antai Ointment can be controlled more efficiently and quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The chromatograms were examined for the extraction solvent ratios of the test samples; S1: methanol: water: n-butyric acid at a volume ratio of 1:1:0.05; S2: methanol: water: n-butyric acid at a volume ratio of 2:1:0.05;

[0030] Figure 2 The chromatograms are used to investigate the extraction time of the test sample; among them, S1: ultrasonic 30min; S2: ultrasonic 40min; S3: ultrasonic 50min;

[0031] Figure 3 It is a specific HPLC chromatogram; among them, S1: negative; S2: gallic acid control; S3: berberine hydrochloride control; S4: ellagic acid control; S5: test sample: 230304;

[0032] Figure 4 It is the precision HPLC chromatogram; Among them, S1-S6: precision 1-precision 6;

[0033] Figure 5 is the repeatability HPLC chromatogram; among them, S1-S6: repeatability 1-repeatability 6;

[0034] Figure 6 It is a stability HPLC chromatogram; among them, S1: 0h; S2: 2h; S3: 4h; S4: 8h; S5: 12h; S6: 24h;

[0035] Figure 7 HPLC chromatograms of eight batches of Antai ointment test products; among them, S1: 230304; S2: 230412; S3: 230516; S4: 230609; S5: 230725; S6: 230823; S7: 231005; S8: 231221;

[0036] Figure 8 This is the reference characteristic spectrum of Antai Ointment. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to the following examples and accompanying drawings. The instruments and equipment used in the examples are as follows:

[0038] 1. Instruments and reagents

[0039] 1.1 Instruments and Equipment

[0040] .

[0041] 1.2 Test materials

[0042] .

[0043] 1.3 Reference Material Information

[0044] .

[0045] 1.4 Sample Information

[0046] .

[0047] Example 1 Investigation of the method for constructing the characteristic spectrum of Antai ointment

[0048] 1.1 Investigation of test sample preparation methods

[0049] 1.1.1 Investigation of extraction solvents for test samples

[0050] 1.1.1.1 Investigation of the extraction solvent ratio of the test sample

[0051] Chromatographic conditions

[0052] Chromatographic column: Inertsustain-C18 (4.6 x 250 mm, 5 μm); use acetonitrile-methanol (27:73) as mobile phase A, dissolve 2.4 g of sodium octane sulfonate in 1000 ml of water, add 4 ml of triethylamine, shake well, and adjust the pH to 3.0 with phosphoric acid to form mobile phase B. Perform gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 250 nm. The number of theoretical plates calculated based on the ellagic acid peak should be no less than 3000.

[0053] .

[0054] Preparation of reference solution

[0055] Take appropriate amount of gallic acid, ellagic acid and berberine hydrochloride reference substances, weigh accurately, add methanol to make a solution containing 60ug per 1ml, shake well, and use it as the reference solution.

[0056] Preparation of test solution

[0057] Take 5 g of Antai Ointment (batch number: 230304), add 50 ml of methanol: water: n-butyric acid in a volume ratio of 1:1:0.05 and 50 ml of methanol: water: n-butyric acid in a volume ratio of 2:1:0.05 respectively, ultrasonically treat for 30 minutes, centrifuge, take the supernatant and evaporate to dryness, add 30 ml of water to dissolve, then add petroleum ether to extract 3 times, 30 ml each time, discard the petroleum ether liquid, and extract the water liquid with n-butanol 3 times, 30 ml each time, evaporate the n-butanol liquid to dryness, add 5 ml of methanol to dissolve, pass through a 0.45 μm microporous filter membrane, discard the initial filtrate, and collect the subsequent filtrate as the test solution.

[0058] Assay

[0059] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0060] The RSDs of the relative retention times of the solvent investigation results were all less than 1.0%, indicating that when the extraction solvents were methanol: water: n-butyric acid at a volume ratio of 1:1:0.05 and methanol: water: n-butyric acid at a volume ratio of 2:1:0.05, the relative retention times of the peaks in the chromatogram were less affected; when methanol: water: n-butyric acid at a volume ratio of 1:1:0.05 was used as the extraction solvent, the peak shapes and separations of the peaks were better. The results are shown in the attached figure. Figure 1 .

[0061] Table 1 Results of relative retention time of solvent investigation

[0062] .

[0063] 1.1.1.2 Investigation of the composition of the test sample extraction solvent

[0064] Chromatographic conditions

[0065] Chromatographic column: Inertsustain-C18 (4.6 x 250 mm, 5 μm); use acetonitrile-methanol (27:73) as mobile phase A, dissolve 2.4 g of sodium octane sulfonate in 1000 ml of water, add 4 ml of triethylamine, shake well, and adjust the pH to 3.0 with phosphoric acid to form mobile phase B. Perform gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 250 nm. The number of theoretical plates calculated based on the ellagic acid peak should be no less than 3000.

[0066] .

[0067] Preparation of reference solution

[0068] Take appropriate amount of gallic acid, ellagic acid and berberine hydrochloride reference substances, weigh accurately, add methanol to make a solution containing 60ug per 1ml, shake well, and use it as the reference solution.

[0069] Preparation of test solution

[0070] Take 5 g of Antai Ointment (batch number: 230304), add 50 ml of methanol: water: n-butyric acid in a volume ratio of 1:1:0.05 and 1:1 volume ratio of methanol: water, respectively, ultrasonically treat for 30 min, centrifuge, evaporate the supernatant, add 30 ml of water to dissolve, then add petroleum ether to extract 3 times, 30 ml each time, discard the petroleum ether liquid, extract the water liquid with n-butanol 3 times, 30 ml each time, evaporate the n-butanol liquid to dryness, add 5 ml of methanol to dissolve, pass through a 0.45 μm microporous filter membrane, discard the initial filtrate, and collect the subsequent filtrate as the test solution.

[0071] Assay

[0072] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0073] The results of the solvent composition investigation showed that when n-butyric acid was present in the extraction solvent, the chromatogram presented 5 characteristic peaks, and the peak shape and separation of each characteristic peak were good. When methanol: water with a volume ratio of 1:1 was used as the extraction solvent, there was a problem of peak missing, so the solvent selected was methanol: water: n-butyric acid with a volume ratio of 1:1:0.05.

[0074] Table 2 Results of relative retention time of solvent investigation

[0075] .

[0076] 1.1.2 Investigation of test sample extraction time

[0077] Preparation of test solution

[0078] Take 5 g of Antai ointment (batch number: 230304), add 50 ml of a mixed solution of methanol: water: n-butyric acid in a volume ratio of 1:1:0.05, ultrasonically treat for 30 min, 40 min, and 50 min, centrifuge, evaporate the supernatant, add 30 ml of water to dissolve, then add petroleum ether to extract 3 times, 30 ml each time, discard the petroleum ether liquid, and extract the water liquid with n-butanol 3 times, 30 ml each time, evaporate the n-butanol liquid to dryness, add 5 ml of methanol to dissolve, pass through a 0.45 μm microporous filter membrane, discard the initial filtrate, and collect the subsequent filtrate as the test solution.

[0079] The chromatographic conditions are the same as those in item “1.1.1”; the reference solution is the reference solution in item “1.1.1”.

[0080] Assay

[0081] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0082] The RSD of the relative retention time of the extraction time investigation results were all less than 1.0%, indicating that the ultrasonic extraction time within 30-50 minutes had little effect on the relative retention time of each peak in the chromatogram; when the ultrasonic extraction time was 30 minutes, the peak shape and separation degree of each peak were better, as shown in the attached results. Figure 2 .

[0083] Table 3 Results of relative retention time during extraction time

[0084] .

[0085] 1.2 Investigation of chromatographic conditions

[0086] 1.2.1 Investigation of mobile phase types

[0087] Chromatographic conditions 1: Inertsustain-C18 (4.6 x 250 mm, 5 μm); acetonitrile-methanol (27:73) as mobile phase A, dissolve 2.4 g of sodium octane sulfonate in 1000 ml of water, add 4 ml of triethylamine, shake well, adjust the pH to 3.0 with phosphoric acid to form mobile phase B, and perform gradient elution as specified in the table below; column temperature 30°C; detection wavelength 250 nm. The theoretical plate number calculated based on the ellagic acid peak should be no less than 3000.

[0088] .

[0089] Chromatographic Conditions 2: Inert Sustain-C18 (4.6 x 250 mm, 5 μm); acetonitrile as mobile phase A, 2.4 g of sodium octane sulfonate dissolved in 1000 ml of water to form mobile phase B, gradient elution as specified in the table below; column temperature 30°C; detection wavelength 250 nm. The theoretical plate number calculated based on the ellagic acid peak should be no less than 3000.

[0090] .

[0091] Chromatographic Conditions 3: Inert Sustain-C18 (4.6 x 250 mm, 5 μm); use methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, using gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 250 nm. The theoretical plate number (theoretical plate number) calculated based on the ellagic acid peak should be no less than 3000.

[0092] .

[0093] Preparation of reference solution

[0094] Take appropriate amount of gallic acid, ellagic acid and berberine hydrochloride reference substances, weigh accurately, add methanol to make a solution containing 60ug per 1ml, shake well, and use it as the reference solution.

[0095] Preparation of test solution

[0096] Take 5 g of Antai Ointment (batch number: 230304), add 50 ml of a mixed solution of methanol: water: n-butyric acid in a volume ratio of 1:1:0.05, ultrasonically treat for 30 minutes, centrifuge, evaporate the supernatant, add 30 ml of water to dissolve, then add petroleum ether to extract 3 times, 30 ml each time, discard the petroleum ether liquid, and extract the water liquid with n-butanol 3 times, 30 ml each time, evaporate the n-butanol liquid to dryness, add 5 ml of methanol to dissolve, pass through a 0.45 μm microporous filter membrane, discard the initial filtrate, and collect the subsequent filtrate as the test solution.

[0097] Assay

[0098] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0099] The results showed that when acetonitrile-methanol (27:73) was used as mobile phase A, 2.4 g of sodium octane sulfonate was dissolved in 1000 ml of water, 4 ml of triethylamine was added, shaken, and the pH value was adjusted to 3.0 with phosphoric acid as mobile phase B for elution, the chromatogram of the test sample showed 5 characteristic peaks, with good peak separation and excellent peak shape; when acetonitrile was used as mobile phase A, 2.4 g of sodium octane sulfonate was dissolved in 1000 ml of water as mobile phase B or when methanol was used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B for elution, the peak shape was poor and peaks were missing, and the corresponding effect was not achieved.

[0100] Table 4 Relative retention time results of mobile phase type investigation

[0101] .

[0102] 1.2.2 Investigation of mobile phase ratio

[0103] Chromatographic conditions: Inertsustain-C18 column (4.6 x 250 mm, 5 μm); acetonitrile-methanol (27:73) as mobile phase A; dissolve 2.4 g of sodium octane sulfonate in 1000 ml of water, add 4 ml of triethylamine, shake well, and adjust the pH to 3.0 with phosphoric acid to form mobile phase B. Gradient elution was performed as specified in the table below; column temperature was 30°C; detection wavelength was 250 nm. The number of theoretical plates calculated based on the ellagic acid peak should be no less than 3000.

[0104] Mobile phase ratio 1:

[0105] .

[0106] Mobile phase ratio 2:

[0107] .

[0108] Mobile phase ratio 3:

[0109] .

[0110] Preparation of reference solution

[0111] Take appropriate amount of gallic acid, ellagic acid and berberine hydrochloride reference substances, weigh accurately, add methanol to make a solution containing 60ug per 1ml, shake well, and use it as the reference solution.

[0112] Preparation of test solution

[0113] Take 5 g of Antai Ointment (batch number: 230304), add 50 ml of a mixed solution of methanol: water: n-butyric acid in a volume ratio of 1:1:0.05, ultrasonically treat for 30 minutes, centrifuge, evaporate the supernatant, add 30 ml of water to dissolve, then add petroleum ether to extract 3 times, 30 ml each time, discard the petroleum ether liquid, and extract the water liquid with n-butanol 3 times, 30 ml each time, evaporate the n-butanol liquid to dryness, add 5 ml of methanol to dissolve, pass through a 0.45 μm microporous filter membrane, discard the initial filtrate, and collect the subsequent filtrate as the test solution.

[0114] Assay

[0115] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0116] The results showed that when eluted with a mobile phase ratio of 1, the test sample chromatogram showed a total of 5 characteristic peaks, with good peak separation and excellent peak shape; when eluted with a mobile phase ratio of 2 or a mobile phase ratio of 3, there were poor peak shapes and peaks missing, and the corresponding effect was not achieved.

[0117] Table 5 Results of relative retention time of mobile phase ratio

[0118] .

[0119] Example 2 Antai ointment characteristic spectrum methodological verification

[0120] 2.1 Exclusivity

[0121] Chromatographic conditions

[0122] Chromatographic column: Inertsustain-C18 (4.6 x 250 mm, 5 μm); use acetonitrile-methanol (27:73) as mobile phase A, dissolve 2.4 g of sodium octane sulfonate in 1000 ml of water, add 4 ml of triethylamine, shake well, and adjust the pH to 3.0 with phosphoric acid to form mobile phase B. Perform gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 250 nm. The number of theoretical plates calculated based on the ellagic acid peak should be no less than 3000.

[0123] .

[0124] Preparation of reference solution

[0125] Take appropriate amount of gallic acid, ellagic acid and berberine hydrochloride reference substances, weigh accurately, add methanol to make a solution containing 60ug per 1ml, shake well, and use it as the reference solution.

[0126] Preparation of test solution

[0127] Take 5 g of Antai Ointment (batch number: 230304), add 50 ml of a mixed solution of methanol: water: n-butyric acid in a volume ratio of 1:1:0.05, ultrasonically treat for 30 minutes, centrifuge, evaporate the supernatant, add 30 ml of water to dissolve, then add petroleum ether to extract 3 times, 30 ml each time, discard the petroleum ether liquid, and extract the water liquid with n-butanol 3 times, 30 ml each time, evaporate the n-butanol liquid to dryness, add 5 ml of methanol to dissolve, pass through a 0.45 μm microporous filter membrane, discard the initial filtrate, and collect the subsequent filtrate as the test solution.

[0128] Preparation of negative solution

[0129] Take an appropriate amount of matrix excipients, add 50 ml of a mixed solution of methanol: water: n-butyric acid in a volume ratio of 1:1:0.05, ultrasonically treat for 30 minutes, centrifuge, evaporate the supernatant, dissolve it in 30 ml of water, then add petroleum ether to extract 3 times, 30 ml each time, discard the petroleum ether liquid, extract the water liquid with n-butanol 3 times, 30 ml each time, evaporate the n-butanol liquid to dryness, add 5 ml of methanol to dissolve it, pass it through a 0.45 μm microporous filter membrane, discard the initial filtrate, and collect the subsequent filtrate as the negative solution.

[0130] Assay

[0131] Accurately pipette 10 μl of each of the negative solution, reference solution and test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0132] The results showed that the negative solution had no interference and good specificity. Figure 5 .

[0133] 2.2 Precision

[0134] Take 5 g of Antai Ointment (Batch No.: 230304) and prepare the test solution according to the test solution preparation method under "2.1". Inject 6 times according to the chromatographic conditions under "2.1", record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0135] Table 6 Precision relative retention time results

[0136] .

[0137] Table 7 Precision relative peak area results

[0138] .

[0139] The results showed that the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 1.0%, indicating good precision. The chromatogram is attached. Figure 6 .

[0140] 2.3 Repeatability

[0141] Take 5 g of Antai Ointment (Batch No.: 230304), a total of 6 portions, and prepare the test solution according to the test solution preparation method under "2.1". Inject the sample according to the chromatographic conditions under "2.1", record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0142] Table 8 Repeatability relative retention time results

[0143] .

[0144] Table 9 Repeatability relative peak area results

[0145] .

[0146] The results showed that the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 1.0%, with good repeatability. The chromatogram is attached. Figure 7 .

[0147] 2.4 Stability

[0148] Take 5 g of Antai Ointment (Batch No.: 230304) and prepare the test solution according to the test solution preparation method under "2.1". Inject the sample at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h according to the chromatographic conditions under "2.1". Record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0149] Table 10 Stability relative retention time results

[0150] .

[0151] Table 11 Stability relative peak area results

[0152] .

[0153] The results showed that the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 1.0%, indicating that the solution was stable. The chromatogram is attached. Figure 8 .

[0154] In summary, the characteristic spectrum has good specificity and there is no interference from negative samples, indicating that the characteristic detection method has strong specificity. In the precision test, the relative retention time RSDs are all less than 1.0%, and the relative peak area RSDs are all less than 1.0%, indicating good instrument precision. In the repeatability test, the relative retention time RSDs of each chromatographic peak are all less than 1.0%, and the relative peak area RSDs are all less than 1.0%, indicating good repeatability. In the stability test, the relative retention time RSDs of each chromatographic peak are all less than 1.0%, and the relative peak area RSDs are all less than 1.0%, indicating that the test solution is stable within 24 hours. This method has been well validated.

[0155] Example 3 Construction of the control characteristic spectrum of Antai Ointment

[0156] Chromatographic conditions

[0157] Chromatographic column: Inertsustain-C18 (4.6 x 250 mm, 5 μm); use acetonitrile-methanol (27:73) as mobile phase A, dissolve 2.4 g of sodium octane sulfonate in 1000 ml of water, add 4 ml of triethylamine, shake well, and adjust the pH to 3.0 with phosphoric acid to form mobile phase B. Perform gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 250 nm. The number of theoretical plates calculated based on the ellagic acid peak should be no less than 3000.

[0158] .

[0159] Preparation of reference solution

[0160] Take appropriate amount of gallic acid, ellagic acid and berberine hydrochloride reference substances, weigh accurately, add methanol to make a solution containing 60ug per 1ml, shake well, and use it as the reference solution.

[0161] Preparation of test solution

[0162] Take 5 g of Antai ointment (batch number: 230304), add 50 ml of a mixed solution of methanol: water: n-butyric acid in a volume ratio of 1:1:0.05, ultrasonically treat for 30 minutes, centrifuge, evaporate the supernatant, add 30 ml of water to dissolve, then add petroleum ether to extract 3 times, 30 ml each time, discard the petroleum ether liquid, and extract the water liquid with n-butanol 3 times, 30 ml each time, evaporate the n-butanol liquid to dryness, add 5 ml of methanol to dissolve, pass through a 0.45 μm microporous filter membrane, discard the initial filtrate, and collect the subsequent filtrate as the test solution.

[0163] Assay

[0164] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0165] Chromatograms from eight batches of Antai ointment were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System." Peaks present in the chromatograms of different batches of Antai ointment were selected as common peaks. A reference characteristic spectrum was generated using the average calculation method, and the relative retention time and relative peak area of ​​each common peak were calculated. The results are shown in Tables 12 and 13.

[0166] Table 12 Relative retention time results of eight batches of Antai ointment samples

[0167] .

[0168] Table 13 Relative peak area results of eight batches of Antai ointment samples

[0169] .

[0170] The chromatograms of the eight batches of Antai ointment samples all showed five common peaks, and the RSDs of the relative retention times and relative peak areas of the five common peaks were all less than 1.0%.

[0171] The test sample chromatogram shows five characteristic peaks, of which Peaks 1, 3, and 5 correspond to the peaks of the reference substance of gallic acid, ellagic acid, and berberine hydrochloride, respectively. The peak corresponding to the peak of the reference substance of ellagic acid is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.33 (Peak 1), 0.80 (Peak 2), 1.22 (Peak 4), and 1.49 (Peak 5).

Claims

1. A method for constructing a characteristic spectrum of Antai ointment, characterized in that: The following steps are involved: (1) Preparation of reference solution: Take the reference substance, weigh it accurately, add methanol to make a solution containing 60 μg per 1 ml, shake well, and use it as the reference solution; the reference substances are: gallic acid, ellagic acid, and berberine hydrochloride; (2) Preparation of test solution: Take 5 g of this product, add 50 ml of a mixed solution of methanol: water: n-butyric acid in a volume ratio of 1:1:0.05 to the test sample, ultrasonicate for 30-50 min, centrifuge, evaporate the supernatant after centrifugation, then add 30 ml of water to the evaporated sample to dissolve it, then add petroleum ether to extract 3 times, using 30 ml of petroleum ether each time, discard the petroleum ether liquid, and extract the water liquid with n-butanol 3 times, using 30 ml of n-butanol each time, evaporate the extracted n-butanol liquid to dryness, add 5 ml of methanol to dissolve it, filter the solution through a 0.45 μm microporous filter membrane, discard the initial filtrate, and collect the subsequent filtrate as the test solution; (3) Determination: Inject the reference solution and the test solution into a high performance liquid chromatograph according to the following chromatographic conditions, inject 10 μl of each sample, and record the chromatogram; Chromatographic conditions were as follows: octadecylsilane bonded silica gel as the filler; acetonitrile-methanol (volume ratio: 27:73) as the mobile phase A, sodium octane sulfonate-triethylamine-phosphoric acid solution as the mobile phase B, elution according to the specified gradient; column temperature: 30°C; flow rate: 1 ml / min; UV detection wavelength: 250-270 nm; The elution gradient conditions were: From 0 to 30 min, the volume ratio of mobile phase A:phase B changed from 5:95 to 40:60; From 30 to 45 min, the volume ratio of mobile phase A:phase B was changed from 40:60 to 70:30; From 45 to 48 minutes, the volume ratio of mobile phase A:phase B was changed from 70:30 to 5:95; 48-60 min, mobile phase A:B, volume ratio 5:95; (4) Generate a control characteristic spectrum: Select the chromatographic peaks that exist in the chromatograms of different batches of An Tai Ointment as common peaks, and use the average value calculation method to generate a control characteristic spectrum of An Tai Ointment.

2. The method according to claim 1, characterized in that The sodium octane sulfonate-triethylamine-phosphoric acid solution in step (3) is prepared as follows: 2.4 g of sodium octane sulfonate is dissolved in 1000 ml of water, 4 ml of triethylamine is added, the mixture is shaken, and the pH value is adjusted to 3.0 with phosphoric acid to obtain a sodium octane sulfonate-triethylamine-phosphoric acid solution.

3. The method according to claim 1, characterized in that The detection wavelength of the ultraviolet detector in step (3) is 250 nm.

4. The method according to claim 1, wherein The chromatographic column in step (3) is: Inertsustain-C18 4.6x250mm, 5um.

5. The method according to claim 1, wherein The control characteristic spectrum generated in step (4) includes five common chromatographic peaks, namely: Peak 1 corresponding to gallic acid, Peak 3 corresponding to ellagic acid, and Peak 5 corresponding to berberine hydrochloride; the characteristic peaks use the ellagic acid chromatographic peak as a reference peak, and the relative retention times of the five chromatographic peaks are: Peak 1 is 0.33, Peak 2 is 0.80, Peak 4 is 1.22, and Peak 5 is 1.

49.

6. Application of the Antai Ointment characteristic spectrum obtained according to the method described in any one of claims 1 to 4 in the quality control of the characteristic components of Antai Ointment.

7. Application of the characteristic spectrum of An Tai Ointment obtained according to the method described in any one of claims 1-4 in the production quality control of An Tai Ointment.

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